1.1 Field of the Invention
Present invention relates to the field of biosensors.
1.2 Description of Related Art
Commercial agriculture depends on monitoring of various plant parameters, such as hydration, disease, ripeness, pest invasion, temperature, adequacy of nutrients, and other conditions to achieve successful yields. Since the beginning of agriculture, farmers relied primarily on their intuition and observation in assessing crop and field conditions. In recent decades, growers increasingly utilize various devices, including computerized systems, containing an assortment of sensing capabilities to more precisely follow plant, field, and greenhouse conditions (Wolf, B. (1996) Diagnostic Technique for Improving Crop Production_l , Haworth Press, pp. 185-187). These new developments are continuously leading to optimization of agricultural production through improved planting, water management, and other practices. However, while these new evolving approaches have substantially enhanced phytomonitoring, the existing methods are still cumbersome, imprecise, require complex and expensive equipment and, in many cases, do not provide real-time monitoring of a crop's condition.
An additional challenge of today's industrialized society is environmental contamination. Increasing effects of chemicals in the environment and their toxicity to human and animal health necessitate monitoring of pollutant levels. Common pollutants, among others, include heavy metals (cadmium, arsenic, mercury, etc), phenolic compounds, etc. Environmental analysis is typically carried out in by sampling of the suspected polluted area and later analyzing the samples using sophisticated methods, such as atomic absorption spectrometry, ion chromatography, etc. These are time consuming and expensive methods, which are not always available or feasible in practicality.
Therefore, there is a clear recognized need for an improved and robust phytosensing method capable of providing reliable real-time information.
U.S. Pat. No. 6,701,665 teaches monitoring of natural plant conditions using computerized systems; however, it does not disclose transgenic plants, nor does it contemplate measurement of active light emission or luminescence from plants.
US patent application 2005/0114923 suggests utilizing expression of plant pigments, such as anthocyanins, generally in response to contaminants. However, the application describes phenotypcial changes in plants, such as pigmentation, and does not relate to detecting plant autoluminescence or monitoring of light-emitting plants. The application also does not relate to or disclose genetic engineering of plastids, nor does it disclose computerized monitoring methods.
Patent applications PCT/US2008/009310, 60/953,337 and PCT/US10/25366 describe incorporation of luciferase and luciferin-related genes into plastids. PCT/US 10/25366 contemplates a genetic relay assay for induction of light emission. These documents do not, however, disclose the use of autoluminescent plants as phytosensors, or contemplate monitoring methods.
WO2007136432 contemplates bioluminescent plants containing LUX operon genes. However, it does not disclose the expression of the LUX operon from the plastid genome, nor does it provide suitable methods or vectors to integrate the LUX operon into plastidal genomes. Furthermore, it does not contemplate indirect luciferase pathway activation in plants (e.g., genetic relay assay as in PCT/US10/25366, etc.), thereby describing a different type of transgenic plants, as well as failing to provide for monitoring or survey methods. Similarly, U.S. Pat. No. 7,049,483 contemplates introduction of jellyfish luciferase and its substrate, coelenterazine, biosynthesis machinery into a plant to generate bioluminescent plants. However, it does not contemplate expression the jellyfish luciferase pathway from plastid genomes, does not provide for suitable methods or vectors to integrate these genes into plastidal genomes, and does not contemplate indirect luciferase pathway activation in plants. Finally, this reference does not contemplate the use of bioluminescent plants as phytosensors.
Therefore, a solid and robust system comprising an autoluminescent plant phytosensor and a computerized monitoring system is needed. The present invention provides for light emitting plants, having light emission machinery integrated within their plastidal genome, and a method of monitoring and surveying light emission thereof in order to utilize these plants as biosensors or phytosensors in agricultural and other settings.
The presently disclosed autoluminescent phytosensor (ALPS) plants, also referred as “ALPS plants” or simply as “ALPS”, address this need by providing a simple, inexpensive, real-time monitoring alternative, superior to other biosensor systems. This technology can be widely used and find application in the monitoring of agricultural and horticultural crops, including ornamentals, and in environmental protection. Finally, ALPS can be used in basic plant research to monitor different plant parameters in real time and with high precision. Currently available monitoring systems frequently measure indirect parameters (e.g., CO2 exchange rate (U.S. Pat. No. 6,701,665)), while ALPS produce signals directly in response to specific stimuli (dehydration, pest invasion, etc.). Moreover, while other reporter systems based on direct coupling of protein expression (e.g., GFP) in response to a given stimulus have been contemplated, those have been proven to be impractical. For example, GFP could not be detected using visualization approaches (see Kooshki et al (2003) “Pathogen inducible reporting in transgenic tobacco using a GFP construct”, Plant Science 165:213-219). ALPS provide for practically feasible, real-time and direct response to very specific stimuli. The technology is non-destructive to plants, does not require any external substrates to be sprayed, and can be remotely sensed.
ALPS based on plastid-integrated light emission systems is a radically new concept. In the past, attempts to incorporate complex metabolic pathways into transgenic plants have been hampered by various limitations of genetic engineering technology. Creation of the world's first autoluminescent plant—a living plant organism capable of emitting visible light on its own, without the need for any external chemical or light sources—has been initially reported by us (Krichevsky et al. (2010) “Autoluminescent Plants”, PLoS ONE 5(11):e15461). Here, for the first time, we describe the use of autoluminescent plants as phytosensors that can be used to monitor plant health, pathogen invasion, environmental contamination, and other conditions and stimuli affecting plant growth and development.